The broad goal of this proposal is to identify the molecular mechanisms that underlie ion permeation and channel gating and its regulation in the inositol trisphosphate receptor (InsP3R). The InsP3R is a ubiquitous family of intracellular Ca2+ release channels that participates in the generation of complex Ca2+ signals that regulate many physiological processes. Its intracellular location has generally hindered study of its single channel properties, and its ubiquitous expression has impeded the development of robust systems for recombinant mammalian isoform expression. Our development of nuclear patch electrophysiology and novel cell systems for mammalian InsP3R expression has now enabled detailed single channel electrophysiological studies of wild-type and mutant channels in native endoplasmic reticulum membrane. We propose three specific aims to characterize the molecular mechanisms that contribute to permeation, gating and channel regulation. First, we will define the molecular determinants of InsP3R channel ion permeation. Here we will employ site-directed mutagenesis of residues in the InsP3R, guided by structural similarities to bacterial cation channels and to the homologous ryanodine receptor. Single channel electrophysiology of isolated nuclei from cells stably expressing mutant InsP3R channels will be used to define the mechanisms that govern channel conductance and ion selectivity properties. Second, we will define the molecular determinants of InsP3R channel gating, focusing on three regions of the channel. Site-directed mutagenesis and single channel electrophysiology will determine the roles of the sixth transmembrane helix (TM6) and the TM4-TM5 linker in activation gating. Biochemical and functional studies will also assess the mechanisms and roles of N- and C-terminal interactions. Third, we will define the molecular determinants of cytoplasmic Ca2+ regulation of channel gating, using a combination of electrophysiological and optical imaging approaches. Here, we will determine the roles of a semi-conserved cytoplasmic region and Ca2+ binding to the InsP3 binding domain in [Ca2+] regulation of gating. The results of these studies should provide new insights into the molecular physiology of this ubiquitous family of Ca2+ release channels.